From 2010.igem.org
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Cloning the rocF BioBrick
Aim
The aim of today's experiment is to amplify 6 different fragments for the construction of rocF BioBrick with the help of 6 different Phusion PCR.
Materials and Protocol
Please refer to PCR for Phusion PCR protocol. The details for the 6 PCR reactions are mentioned below:
PCR
| Tube
| Part to be amplified
| Plasmid consisting the part
| Forward primer
| Reverse Primer
| Melting Temperature (Tm in °C)
| Size of the fragment (in bp)
| Extension time* (in seconds)
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| 1
| Plasmid Vector
| pSB1C3
| P1V1 forward
| P2V1 reverse
| 58
| 2072 approx.
| 60
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| 2
| Pspacoid Promoter
| pMutin4
| P1P1 forward
| P2P1 reverse
| 49
| 106 approx.
| 15
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| 3
| 1st fragment of rocF CDS
| B. subtilis 168 chromosome
| P1S1 forward
| P2S1 reverse
| 58
| 246 approx.
| 15
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| 4
| 2nd fragment of rocF CDS
| B. subtilis 168 chromosome
| P3S2 forward
| P4S2 reverse
| 65
| 597 approx.
| 20
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| 5
| 3rd fragment of rocF CDS
| B. subtilis 168 chromosome
| P5S3 forward
| P6S3 reverse
| 66
| 125 approx.
| 15
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| 6
| Double Terminator
| pSB1AK3 consisting BBa_B0014 biobrick
| P1T1 forward
| P2T1 reverse
| 56
| 116 approx.
| 15
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Gibson assembly
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